Interaction between P311 and transforming growth factor beta 1 and its effect on the function of murine fibroblasts
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摘要: 目的 探讨P311和TGF-β1在小鼠皮肤Fb中的相互作用及其对Fb功能的影响。 方法 取P311野生型和P311基因敲除型C57BL/6新生小鼠各5只,分离培养2种小鼠皮肤Fb。采用第2代Fb完成以下实验,每个实验重复3次。(1)取P311野生型小鼠Fb,按随机数字表法(分组方法下同)分为空白对照组和P311过表达组,每组36孔。空白对照组每孔加入10 μL空载体腺病毒,P311过表达组每孔加入等效价P311腺病毒表达载体。培养48 h后,采用实时荧光定量RT-PCR法和蛋白质印迹法分别检测2组Fb的P311 mRNA及TGF-β1、α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原的mRNA和蛋白表达量(检测方法下同)。(2)取P311野生型和P311基因敲除型小鼠Fb各4瓶,待细胞生长至80%~90%融合时,检测2种Fb的TGF-β1、α-SMA、Ⅰ型胶原的mRNA和蛋白表达量。(3)取P311野生型小鼠Fb用体积分数为1%FBS的DMEM培养液饥饿处理3 h后,分为空白对照组及5、10、15、20、25 ng/mL TGF-β1组,每组2瓶。空白对照组常规培养,后5组Fb分别加入5、10、15、20、25 ng/mL TGF-β1溶液,培养48 h后检测6组Fb的P311 mRNA表达量。另取P311野生型小鼠Fb,分为空白对照组和10 ng/mL TGF-β1组,每组6孔,免疫荧光法检测2组Fb的P311蛋白表达情况。(4)将P311野生型小鼠Fb同前饥饿处理后,分为空白对照组和10 ng/mL TGF-β1组,每组2瓶,空白对照组常规培养,后组加入10 ng/mL的TGF-β1溶液,培养48 h后,检测2组Fb的α-SMA、Ⅰ型胶原的mRNA和蛋白表达量。取P311基因敲除型小鼠Fb,同前分组及处理后,检测2组Fb的α-SMA、Ⅰ型胶原的mRNA和蛋白表达量。对数据行单因素方差分析、
t 检验。 结果 (1)P311过表达组Fb的P311 mRNA表达水平较空白对照组增高近30万倍(t =9.942,P <0.001)。P311过表达组Fb的TGF-β1、α-SMA、Ⅰ型胶原的mRNA,以及TGF-β1前体、TGF-β1活化态、α-SMA、Ⅰ型胶原的蛋白表达量均较空白对照组明显升高(t 值为8.192~49.090,P 值均小于0.01)。(2)P311基因敲除型小鼠Fb的TGF-β1、α-SMA、Ⅰ型胶原的mRNA表达量较P311野生型小鼠Fb明显降低(t 值为8.157~22.270,P 值均小于0.01)。P311基因敲除型小鼠Fb的TGF-β1前体、α-SMA、Ⅰ型胶原的蛋白表达量较P311野生型小鼠Fb明显降低(t 值为2.995~12.600,P <0.05或P <0.01),2种Fb的TGF-β1活化态蛋白表达量相近(t =1.070,P >0.05)。(3)空白对照组及5、10、15、20、25 ng/mL TGF-β1组Fb的P311 mRNA表达量分别为1.28±0.44、3.61±0.91、6.64±0.92、6.58±1.04、1.79±0.31、0.16±0.06。与空白对照组Fb的P311 mRNA表达量比较,5、20 ng/mL TGF-β1组无明显变化(t 值分别为2.302、0.955,P 值均大于0.05),10、15 ng/mL TGF-β1组明显增高(t 值分别为5.630、4.710,P 值均小于0.001),25 ng/mL TGF-β1组明显降低(t =2.509,P <0.01)。10 ng/mL TGF-β1组Fb的P311蛋白表达明显强于空白对照组。(4)P311野生型小鼠10 ng/mL TGF-β1组Fb的α-SMA、Ⅰ型胶原的mRNA和蛋白表达量均较空白对照组有不同程度的升高(t 值为3.523~14.290,P <0.05或P <0.01)。P311基因敲除型小鼠10 ng/mL TGF-β1组Fb的α-SMA、Ⅰ型胶原的mRNA和蛋白表达量均较空白对照组有不同程度的升高(t 值为4.895~14.870,P <0.05或P <0.01)。 结论 P311和TGF-β1在小鼠皮肤Fb中存在相互作用,可共同促进Fb的分化。Abstract: Objective To explore the interaction between P311 and transforming growth factor beta 1 (TGF-β1) in murine fibroblasts and its effect on the function of fibroblasts. Methods Skin fibroblasts obtained from five neonatal P311 wild-type C57BL/6 mice and P311 gene knock-out C57BL/6 mice were cultured. The second passage of fibroblasts were used in the following experiments. All experiments were repeated for 3 times. (1) The fibroblasts of P311 wild-type mice were divided into blank control group and P311 over-expression group according to the random number table (the same grouping method below), with 36 wells in each group. Fibroblasts in blank control group were transfected with 10 μL control vector, and fibroblasts in P311 over-expression group were transfected with equal efficiency P311 expression adenovirus vector. After being cultured for 48 hours, the mRNA expression level of P311, and the mRNA and protein expression levels of TGF-β1, α-smooth muscle actin (α-SMA), and collagen type Ⅰ of fibroblasts in both groups were determined with real-time fluorescent quantitative RT-PCR and Western blotting (the same detection methods below), respectively. (2) After cultured reaching the cell density of 80%-90%, the mRNA and protein expression levels of TGF-β1, α-SMA, and collagen type Ⅰ of the fibroblasts of P311 wild-type mice and P311 gene knock-out mice, with 4 flasks in each type of fibroblasts, were determined. (3) The fibroblasts of P311 wild-type mice were divided into blank control group and 5, 10, 15, 20, and 25 ng/mL TGF-β1 groups after being starved treatment with DMEM medium containing 1% FBS for 3 hours, with 2 flasks in each group. Fibroblasts in blank control group were routinely cultured, while fibroblasts in the latter five groups were treated with 5, 10, 15, 20, and 25 ng/mL TGF-β1, respectively. After being cultured for 48 hours, the mRNA expression levels of P311 in fibroblasts of the six groups were determined. Another fibroblasts of P311 wild-type mice were divided into blank control group and 10 ng/mL TGF-β1 group, with 6 wells in each group, and the protein expression levels of P311 in both groups were determined by immunofluorescence staining. (4) The fibroblasts of P311 wild-type mice were divided into blank control group and 10 ng/mL TGF-β1 group after being starved treatment as above, with 2 flasks in each group, and fibroblasts in blank control group were routinely cultured, while fibroblasts in the latter group were treated with 10 ng/mL TGF-β1. After being cultured for 48 hours, the mRNA and protein expression levels of α-SMA and collagen type Ⅰ were determined. The fibroblasts of P311 gene knock-out mice were grouped and treated as above, and the mRNA and protein expression levels of α-SMA and collagen type Ⅰ were determined. Data were processed with one-way analysis of variance andt test. Results (1) The mRNA expression level of P311 of fibroblasts in P311 over-expression group was increased nearly 300 000-fold compared with that in blank control group (t =9.942,P <0.001). The mRNA expression levels of TGF-β1, α-SMA, and collagen type Ⅰ of fibroblasts in P311 over-expression group, and the protein expression levels of pro-TGF-β1, activated TGF-β1, α-SMA, and collagen type Ⅰ of fibroblasts in P311 over-expression group were significantly higher than those in blank control group (witht values from 8.192 to 49.090,P values below 0.01). (2) The mRNA expression levels of TGF-β1, α-SMA, and collagen type Ⅰ in fibroblasts of P311gene knock-out mice were significantly lower than those in fibroblasts of P311 wild-type mice (witht values from 8.157 to 22.270,P values below 0.01). The protein expression levels of pro-TGF-β1, α-SMA, and collagen type Ⅰ in fibroblasts of P311 gene knock-out mice were significantly lower than those in fibroblasts of P311 wild-type mice (witht values from 2.995 to 12.600,P <0.05 orP <0.01), and the protein expression levels of active TGF-β1 were similar in two types of fibroblasts (t =1.070,P >0.05). (3) The mRNA expression levels of P311 of fibroblasts in blank control group and 5, 10, 15, 20 and 25 ng/mL TGF-β1 groups were 1.28±0.44, 3.61±0.91, 6.64±0.92, 6.58±1.04, 1.79±0.31, 0.16±0.06, respectively. Compared to the mRNA expression level of P311 of fibroblasts in the blank control group, the mRNA expression levels of P311 of fibroblasts in 5 and 20 ng/mL TGF-β1 groups were similar (witht values respectively 2.302 and 0.955,P values above 0.05), while they were significantly higher in 10 and 15 ng/mL TGF-β1 groups (witht values respectively 5.630 and 4.710,P values below 0.001), and they were significantly lower in 25 ng/mL TGF-β1 group (t =2.509,P <0.01). The protein expression level of P311 of fibroblasts in 10 ng/mL group was higher than that in blank control group. (4) The mRNA and protein expression levels of α-SMA and collagen type Ⅰ of fibroblasts of P311 wild-type mice in 10 ng/mL TGF-β1 group were significantly higher than those in blank control group (witht values from 3.523 to 14.290,P <0.05 orP <0.01). The mRNA and protein expression levels of α-SMA and collagen type I of fibroblasts of P311 gene knock-out mice in 10 ng/mL TGF-β1 group were significantly higher than those in blank control group (witht values from 4.895 to 14.870,P <0.05 orP <0.01). Conclusions The interaction between P311 and TGF-β1 in murine fibroblasts exists and it may enhance the differentiation of fibroblasts in combination.-
Key words:
- Cicatrix /
- Fibroblasts /
- Transforming growth factor beta1 /
- P311
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